Modulation of glucagon receptor expression and response in transfected human embryonic kidney cells

The modulation of glucagonreceptor (GR) expression and biological response was investigated inhuman embryonic kidney cell (HEK-293) clones permanently expressing theGR with different densities. The GR mRNA expression level in theseclones was upregulated by cellular cAMP accumulation and presented agood correlation with both the protein expression level and the maximumnumber of glucagon binding sites. However, the determination ofglucagon-induced cAMP accumulation in these cell lines revealed thatthe enhancement of receptor expression did not lead to a proportionalincrease in cAMP formation. Under these conditions, the maximumcAMP production induced by NaF and forskolin was not significantlydifferent among selected clones, regardless of the receptor expressionlevel. High receptor-expressing clones showed the greatestsusceptibility for agonist-induced desensitization compared with cloneswith lower GR expression levels. The results of the present studysuggest that the GR can recruit non-GR-specific desensitizationmechanism(s). Furthermore, the partial inhibition or alteration of theoverall cAMP synthesis pathway at the receptor level may be a necessary adaptive step for a cell in response to a massive increase in membranereceptor expression level.

     

Over-expression of wild-type and mutant HFE in a human melanocytic cell line reveals an intracellular bridge between MHC class I pathway and transferrin iron uptake

Hereditary hemochromatosis (HH) is a frequent recessive disorder of iron metabolism characterised by systemic iron overload. In Northern Europe, more than 90% of HH patients are homozygous for a mis-sense mutation (C282Y) in the HFE1 gene product. The HFE protein is the heavy chain of a MHC class I-related molecule and associates with beta2 microglobulin and the transferrin receptor. Its precise roles in iron metabolism and in the pathophysiology of HH are still unclear. In order to identify the cellular processing of HFE, an important step towards the understanding of the function of the protein, we stably over-expressed the wild type and mutated forms fused to the Green Fluorescent Protein in a melanocytic MHC class I expressing cell line, the Mel Juso cell line. In wild type and mutant clones, the fusion proteins were not detected at the cell surface but only in the cytoplasm. Their sub-cellular localisation was determined by co-labelling of cells with organite-specific antibodies and confocal microscopy. HFE-GFP followed initially HLA class I intracellular processing but co-localised with transferrin in early endosomes without recycling at the cell surface. The C282Y-GFP fusion protein followed a different folding pathway to exit endoplasmic reticulum. Over-expression of the wild-type protein lead to a decrease in diferric transferrin uptake. Our model will be of use in the elucidation of the functional interaction between intracellular HFE and iron transporters transferrin/transferrin receptor complexes and Slc11A2 (also named N-Ramp2 or DMT1) in different endosomal compartments.     

Nerve growth factor expression response to induction agent booster dosing in transfected human embryonic kidney cells

To assess whether nerve growth factor (NGF) expression would respond to booster dosing with the inducing agent ponasterone A, human embryonic kidney cells (HEK-293) were transfected with human NGF cDNA. Cells were cultured for 5 days in media with or without ponasterone A. On day 5, controls received a ponasterone A media replacement, whereas experimental groups received ponasterone A booster media replacement. NGF protein expression bioactivity was assessed using a PC-12 cell bioassay and the concentration of secreted NGF was quantified using NGF enzyme-linked immunosorbent assay. Cells with and without ponasterone A were left for 5 days without changing the medium. On day 5, the supernatants were collected and flash-frozen for enzyme-linked immunosorbent assay. The ponasterone A-positive and -negative booster medium was replaced in the appropriate wells. Supernatants were collected from the wells at 2, 4, and 6 days after the booster dose and removal of original supernatant. The medium was flash-frozen for enzyme-linked immunosorbent assay (1.5 ml), and the remaining 500 mul was transferred to PC-12 cells seeded onto 12-well plates to determine NGF bioactivity. All experiments were performed in quadruplicate. NGF production was measured daily by enzyme-linked immunosorbent assay over a 6-day period after the ponasterone A booster to a maximal release of 1233 +/- 130 pg/ml at day 6 (11 days after original induction). Maximal NGF production per 10(3) cells was 2.5 +/- 0.61 pg at day 6. Bioactivity was determined by percentage differentiation (per 100 cells counted) at 26, 52, and 98 percent for ponasterone A-treated wells on 2, 4, and 6 days after booster dosing (7, 9, and 11 days after induction), respectively. PC-12 cell differentiation was not visualized in the ponasterone A-negative control wells. Human NGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent ponasterone A. Furthermore, repeated bioactive NGF expression peaks beyond that previously demonstrated can be achieved using induction agent booster dosing, indicating the ability to regulate the system over an extended period.     

Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

The hereditaryhemochromatosis protein HFE is known to complex with the transferrinreceptor; however, its function regarding endocytosis of transferrin isunclear. We performed patch-clamp capacitance measurements intransfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNAunder the control of a tetracycline-sensitive promoter. Whole cellexperiments in cells with suppressed expression of wild-type HFErevealed a decrease in membrane capacitance, reflecting predominance ofendocytosis in the presence of transferrin. Cells overexpressingC282Y-mutant HFE displayed less intense capacitance decreases, whereasno significant decrease was observed in cells overexpressing wild-typeHFE. The formation of single endocytic vesicles in cells withsuppressed expression of wild-type HFE was greatly increased in thepresence of transferrin as revealed by cell-attached recordings.According to their calculated diameters, many of these vesiclescorresponded to clathrin-coated vesicles. These results suggest thatwild-type HFE negatively modulates the endocytic uptake of transferrin.This inhibitory effect is attenuated in cells expressing C282Y-mutantHFE. Time-resolved measurements of cell membrane capacitance provide apowerful tool to study transferrin-induced endocytosis in single cells.

     

Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells

X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI(-)), and was recently used in our 2.1-? X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ～0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8-10 weeks.     

HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron

Mutations in either HFE or transferrin receptor 2 (TfR2) cause decreased expression of the iron regulatory hormone hepcidin and hemochromatosis. HFE and TfR2 were recently discovered to form a stable complex at the cell membrane when co-expressed in heterologous cell lines. We analyzed the functional consequences of the co-expression of these proteins using transfected TRVb cells, a Chinese hamster ovary derived cell line without endogenous HFE or transferrin receptor. The co-expression of TfR2 in TRVb cells expressing HFE led to accelerated HFE biosynthesis and late-Golgi maturation, suggesting interaction prior to cell surface localization. The co-expression of HFE in cells expressing TfR2 led to increased affinity for diferric transferrin, increased transferrin-dependent iron uptake, and relative resistance to iron chelation. These observations indicate that HFE influences the functional properties of TfR2, and suggests a model in which the interaction of these proteins might influence signal transduction to hepcidin.     

Transactivation properties of wild-type and mutant androgen receptors in transiently transfected primary human fibroblasts

BACKGROUND: Stromal cells play key roles during androgen-mediated male sexual differentiation. Our objective was to establish a transient transfection method for primary human fibroblasts enabling functional characterization of wild-type (wt) and mutant androgen receptor (AR) plasmid constructs, corresponding to partial and complete androgen insensitivity syndrome (PAIS/CAIS). METHODS: An AR-negative fibroblast strain (ARD842) was established from the gonads of a CAIS patient. Wt-AR or either mutants L712F (PAIS), R774C or V866M (CAIS) were transfected using a polyamine-based procedure. Alternatively, two AR-positive male foreskin fibroblast strains were investigated. Androgen-induced activation of two co-transfected reporter plasmids ((ARE)(2)TATA-, MMTV-luciferase) was measured. RESULTS: All three fibroblast strains showed a ligand-dependent rise of luciferase activity after transfection of wt-AR. Mutant plasmids were assessed in AR-negative ARD842 cells. While L712F showed high partial activity, R774C and V866M were nearly inactive. The intrinsic AR of normal foreskin fibroblasts revealed no measurable ligand-inducible reporter gene activity. CONCLUSIONS: Polyamine-based transfection of AR plasmids into cultured fibroblasts provides a promising tool for analysis of AR transactivation, thereby considering a stromal cellular background. This is supported by the mutant ARs which showed the expected levels of impaired transactivation with respect to the corresponding AIS phenotypes. The role of the intrinsic AR in normal male human foreskin fibroblasts needs further exploration.     

Shear stress effects on human embryonic kidney cells in Vitro

Human embryonic kidney cells grown as an attached, confluent monolayer on a flat substrate were subjected to steady, uniform laminar flow of medium in a specially designed chamber in which flow patterns and shear stress are accurately defined and controlled. Experiments were performed for shear stress levels ranging from 0.2 to 6.0 N/m(2) with times of exposure to the shear stress ranging from 2 to 24 h. The influence of the shear field was slight at low shear stress (0.26 N/m(2)). Higher stress levels (0.65 N/m(2) and higher) had significant effects on cell morphology, and on the post-shear release of urokinase enzyme. Still higher stress levels (2.6 N/m(2) and higher) caused marked reduction in cell viability. These results may be of interest in addressing practical problems in developing commercial biosynthesis reactors.     

The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells

HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C., Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E., Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B., Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana, L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna, D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996) Nat. Genet. 13, 399-408). At the cell surface, HFE complexes with transferrin receptor (TfR), increasing the dissociation constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998) J. Biol. Chem. 273, 22068-22074; Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N. , Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A 95, 1472-1477). HFE does not remain at the cell surface, but traffics with TfR to Tf-positive internal compartments (Gross et al., 1998). Using a HeLa cell line in which the expression of HFE is controlled by tetracycline, we show that the expression of HFE reduces 55Fe uptake from Tf by 33% but does not affect the endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to reduce cellular acquisition of iron from Tf within endocytic compartments. HFE specifically reduces iron uptake from Tf, as non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not altered. These results explain the decreased ferritin levels seen in our HeLa cell system and demonstrate the specific control of HFE over the Tf-mediated pathway of iron uptake. These results also have implications for the understanding of cellular iron homeostasis in organs such as the liver, pancreas, heart, and spleen that are iron loaded in hereditary hemochromatotic individuals lacking functional HFE.     

Interaction of hemojuvelin with neogenin results in iron accumulation in human embryonic kidney 293 cells

Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77-82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis.     

Connexin mutant embryonic stem cells and human diseases

Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.     

Proinsulin-like growth factor-II overexpression does not alter monoallelic H19 gene expression in transfected human embryonic kidney fibroblasts

Insulin-like growth factor-II (IGF-II) is a potent mitogen for cells in culture. The H19 gene is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in tumorigenesis. The H19 gene is closely linked to the human IGF-II gene (IGF2) on chromosome 11p15.5 and these genes are reciprocally imprinted in most fetal tissues. H19 is expressed only from the maternal and IGF2 from the paternal chromosome. We have asked whether overexpression of proIGF-II alters H19 imprinting status and/or expression. Human embryonal kidney fibroblasts (293 cells) were stably transfected with a PCMV5 vector containing the full length human IGF-II cDNA or a control cDNA. Transfectant clones expressed large quantities of IGF-II mRNA and secrete 1-5 ug/ml and 150-230 ng/ml proIGF-II within 24 hours of serum-free culture (transfectant 293-9 and -11 respectively) (1). Cells were genotyped at the exon 5, RsaI restriction fragment length polymorphism (RFLP) and found to be informative (+/-). H19 expression was monoallelic (+) indicating preservation of H19 imprinting in all cell lines. Using quantitative RT-PCR with internal competitors for H19 and for IGF-II cDNA, overexpression of IGF2 in 293-11 and 293-9 cells was confirmed. In contrast, no significant difference with respect to H19 expression was detected between the overexpressing cells and control lines. In conclusion, (1) human embryonal fibroblasts express the H19 gene. (2) H19 imprinting is preserved in these cells. (3) proIGF-II overexpression does not alter H19 expression.     

Butyrate induces expression of transfected human fetal and endogenous mouse embryonic globin genes in GM 979 erythroleukemia cells

We have analyzed the expression of endogenous murine genes and of transfected human fetal A gamma globin gene in GM 979, a mouse erythroleukemia line which produces adult as well as embryonic globins. Optimal induction of the endogenous murine adult globin genes was obtained with DMSO or HMBA while the epsilon y and beta h1 embryonic genes were preferentially induced by butyrate. Similarly, the transferred human A gamma-globin gene was preferentially induced by butyrate. These results as well as previous observations in vivo or in erythroid cell cultures suggest that butyrate preferentially induces the expression of fetal globin genes.